DEGRADATION AND

PHYTOTOXICITY OF
BIODIESEL OIL
Contract Ref: CSA 2614

Caroline Birchall, Jonathan R. Newman & Michael P. Greaves

lnsritute of Arable Crops Research

Long Ashton Research Station,
CENTRE FOR AQUATIC PLANT MANAGEMENT
Broadmoor Lane,
Sonning-on-Thames,
Reading.
RG4 OTH.
Tel.: (01734)
Fax.: (01754)

690072
441730

CONTENTS

Page

CONTENTS

LIST OF TABLES

ii

1.

EXECUTIVE SUMMARY

2.

INTRODUCTION

2.1
2.2

Background
The Research

3
3

TOXlCirY TO ALGAE

3.1
3.2

4
5

3.

4.

5.

TCXlCl-iY TO FLOATING MACROPHYTES

4.1
4.2

8
9

8.

12

Materials and Methods

Results and Discussion

12
13

TOXICITY TO INVERTEBRATES

17

Materials and Methods

Results and Discussion

77
18

TOXICITY IN AQUATIC MICROCOSMS

21

7.1
7.2

Materials and Methods

Results and Discussion

21
22

BIODE,GRADATION AND FATE

24

8.1
8.2

24
26

6.1
6.2
7.

Materials and Methods

Results and Discussion

T0XlCll-Y TO SUBMERGED MACROPHYTES

5.1
5.2

6.

Materials and Methods

Results and Discussion

Materials and Methods

Results and Discussion

9.

GENERAL CONCLUSIONS

28

10.

REFERENCES

29

11.

APPENDICES

30

APPENDIX 1.

APPENDIX 2.

Temperature, pH and Dissolved Oxygen

Concentrations in Aquatic Microcosms

30

Eiodiesel Spectra and Ion Chromatograms

35

APPENDIX

3.

Diesel Spectra and Ion Chromatograms

40

APPEN D IX

4.

Biodiesel and Diesei Ion Spectra Library Searches

44

A P P E N D IX

5.

Mass Spectrometer and Gas Chromatography Instruments

4 9

List of Suppliers

50

APPENDIX 6.

LIST OF TABLES
1.

The effect of biodiesel and marine diesel on the growth of freshwater algae

2.

The effect of biodiesel and marine diesel on specific growth rates of freshwater algae

3.

The effect of biodiesel and marine diesel on growth of Lemna minor and Lemna minuta

4.

The effect of biodiesel and marine diesel on growth of Lemna minor and Lemna minuta

70

5.

The growth measured as the increase in the number of green fronds, of Lemna minor
and Lemna minufa on 7 day old medium contaminated with biodiesel and marine diesel

11

The effect of biodiesel and marine diesel applied to the water suriace on the growth
of Elodea canadensis and Myriophyllum spicatum.

13

The effect of biodiesel and marine diesel applied to the water surface on the growth
of Eiodea canadensis and Myriophyllum spicatum.

14

6.

7.

a.

The effect of biodiesel and marine diesel applied to the hydrosol on the growth
of Eiodea canadensis and Myriophyllum spicatum.

9.

The effect of biodiesel and marine diesel applied to the hydrosol on the growth
of Elodea canadensis and Myriophyllum spicatum.

15

IO.

The effect of biodiesel and marine diesel on Daphnia magna.

ia

11.

The effect of biodiesel and marine diesel on Gammarus pulex.

19

12.

The effect of biodiesel and marine diesel on Lymnaea peregra.

19

13.

The mean 10s of weight of fish in aquatic microcosms contaminated with biodiesel
and marine diesel oil.

23

14.

Partition of biodiesel and marine diesel into aquatic microcosm components.

26

15.

Persistence of biodiesel and marine diesel in microcosms.

27

ii

1.

..

EXECUTIVE SUMMARY

A short pre!iminary examination has been made of the comparative toxicity of rape methyl ester (biodiesel)
and marine diesel to a range of aquatic species. Partitioning and persistence of the oils in aquatic
microcosms have also been examined. The oils were tested at a range of dose rates calculated to represent
light, medium and severe spillages.
Bearing in mind that the data from short duration acute toxicity tests with small numbers of replicates are
not unequivocal the following general conclusions appear warranted. 1

Biodiesel is considerably less toxic to microalgae (green and blue-green species) than marine diesel
even at high dose rates.

The floating plant, Lemna minor (Duckweed) was affected equally by both oiis, growth being reduced
by 65% at the highest doses. The related Lemna minuta, however, was significantly more
susceptibie to marine diesel, the highest dose killing the plant whereas biodiesel merely reduced its
growth by 60%. The susceptibility of both species was increased somewhat when nutrient
concentrations were reduced but still grew in the highest doses of biodiesel but not in marine diesel.
Submerged macrophytes grew erratically in the tests and produce imprecise data. Even so
Myriophyllum spicarum (Water milfoii) was clearly much more susceptible to marine diesel than
biodiese!. Nodea canadensis (Canadian pondweed) appeared to be severely affected by low doses
of biodiesel but less so by higher doses. Marine diesel was severely toxic at moderate to high
doses.
When biodiesel was applied to the sediments it appeared to stimulate growth of E. canadensis the
effect increasing with dose. Marine diesel was increasingly toxic to this species with increasing
dose. M. spicatum unusually was inhibited by both low and high doses of both oils but unaffected
by medium doses.

The invertebrates Daphnia magna (water flea), Gammarus puiex (water louse) and Lymnea peregra
(water snai) were highly sensitive to marine diesel, all animals being killed relatively quickly at all
doses. D. magna and L. peregra were much more tolerant of biodiesel, effects only being severe
at the highest dose. G. pulex was more sensitive, mortality being high even at relatively low doses.

P.ssays of toxicity to mixed species were inconclusive, mainly due to the effects of the unusually high
ambient temperatures raising water temperatures during the tests. Observations suggest that
rainbow trout, Onchorhynchus mykill, were more severely affected by marine diesel than biodiesel.
Body weight loss was greater and the fish showed more severe behavioural symptoms, loss of
baiance, muscular spasms and erratic fin and gill movements.

Biodiesel forms discrete globules on the water sunace whereas marine diesei produced a continuous
slick that is potentially more damaging to invertebrates moving or breathing at the water suriace.
Eoth oils entered the water column from surface deposits, and contaminated plants and sediments,
very quickly in the microcosms. This is likely to be aided by the water agitation resulting from forced
ae:ation which may, to some extent, mimic the result of boat propeller action.
In the conditions of the test biodiesel formed waxy deposits on leaf sunaces, possibly by interaction
with calcium carbonates produced at the leaf surface during the elevated respiration induced by high
water temperatures.

Biodiesel. as measured by GC/MS analysis of a major characteristic methyl ester ion, disappeared
from the water body, plants and sediments significantly more quickly than marine diesel. There was
no evidence of persistent intermediate compounds and it is likely that all the oii was rapidly
degraded to CO,.
7

The foregoing suggests that biodiesel is generally less toxic than marine diesel and persists less.
The toxicity it does exert may, especially at high contamination rates, cause some shifts in species
Overall, however, biodiesel does appear to offer considerable
balance for a short time.
environmental advantage over marine diesel as a boat fuel, especially in iniand waterJJays of
conservation value. This conclusion needs confirmation by more detailed and precise examinations
of ecotoxicity, persistence, and fare.

2.

INTRODUCTION

2.7

Background

Agriculture has the potential to produce a large number of renewable fuels. The most obvious is
wood, but any plant tissue can be used, either directly or after processing, as a biofuel .
in recent years there has been growing interest in Europe in the use of modified Rapeseed oil as
a diesel substitute. Rapeseed Oil Methyl Ester (RME), commonly known in Britain as biodiesel, is
produced by a simple esterification process:

5ooc
RAPESEED OIL + methanol

p
NaOH

diester (RME) + glycerol

A new approach to RME production is currently under investigation to determine if methanol, of

fossil fuel origin, can be replaced by ethanol, produced from biomass. This would result in a
biofuel produced entirely from renewable resources.
Using the whole of the UKs set-aside land (1/6th of the arable land in the UK) to produce
rapeseed would supply only 6% of the UK current diesel fuel requirements, so there is little
possibility of biodiesel completely replacing conventional diesel. However, because vegetable oils
such as rapeseed oil are more readily biodegradable than mineral oils, biodiesei could give
environmental benefits in niche fuel markets such as National Parks and inland waterways. There
is a particular concern that water pollution by conventional diesel fuel is causing severe
environmental problems in many inland waterways, especially those such as the Norfolk Broads
which are exposed to heavy boat trafiic.
2.2

The Research

This study was established as a prelininary examination of the comparative toxicity of diesel and
biodiesel fuels to a range of aquatic species at doses which might result from spillages from boats.
The partition of the fuels in the aquatic system and their degradation are also examined.
Two specific objectives are addressed:
A.

To assess the toxicity to algae, macrophytes and invertebrates, using single-species

toxicity tests

To determine the partition and degradation in water, sediment and plants determined in
aquatic microcosms

3.

TOXICITY TO ALGAE

3.1

Materials and Methods

The toxicity of biodiesel and marine diesel to unicellular freshwater algae was tested in standard 4 day
laboratory bioassays. (ASTM, 1993).
The algal culture medium used was Jaworskis medium (JMl) CThompson et al., 1988). This was prepared
by mixing 1 ml of each of the following stock solutions and making up to 1 litre with glass distilled water.
per ZOOmI
4.0000 g
2.4800 g
10.0000 g
3.1800 g
0.4500 g
0.4500 g
0.4960 g
0.2780 g
0.2000 g
0.0008 g
0.0008 g
0.0008 g
16.0000 g
7.2000 g

Ca(N0,),.4H,O
KH,PO,
MgSO,.7H,O
NaHCO,
EDTA FeNa
EDTA Na,
H,BO,
MnC1,.4H,O
(NH,),Mo,%4H,C
Cyanocobalamin (Viiamin 612)
Thamine HCl (Vitamin Bl)
Biotin
NaNO,
Na,HPO,.l2H,O

The pH was corrected to pH 7.5 with 0.1 M HCI and buffered using 50 mmol HEPES. 50 ml of culture
medium was added to 21 x 250 ml conical flasks. The fiasks were autoclaved at 121C, 15 bar, for 15
minutes
The flasks were
and a l l o w e d t o c o o l t o room t e m p e r a t u r e .
innoculated with 1 ml algal culture. The mean number of cells per ml in the innocula (3 replicates) was
determined using an Improved Neubauer Haemocytometer. The flasks were incubated on an orbital shaker
at 50 rpm at 20 IlC at an incident irradiance of 160 p mol photons m s- supplied by a bank of twelve 36W
fluorescent tubes.
Oils were added aseptically to the cultures using micropipettes after growth for 24 h,, to give dose rates of,
1, 10 and 100 g/l. These rates simulate minimal, average and gross spillage. Chiorella vulgaris was found
in a preliminary experiment to be relatively tolerant of both oils and it was exposed to dose rates of 2, 20
and 200 g/l. Each dose rate and the oil-free controls were replicated 3 times. After the oil was applied the
flasks were incubated for a further 72 hours, after which ceil numbers were determined using the
haemocytometer method.
The algal species examined were Chlorella vulgaris Beijererinck 1890 (Division Chlorophyta, Order
Chlorococcaies, Family Oocystaceae); Microc;/stk aeruginosa KiXng emend. Elenkin 1924, (Division
L ceae); Anabaena spiroides Klehbahn 1895 (Division
Cyanophyta, Order Chroococcaies, Family Chroococ-a
Cyanophyta, Order Osciilatoriales, Family Nostccsceae); Selenastrum ca,@cornutum Prinz 1913 (Division
Chlorophyta. Order Chlorococcales, Family Scenedesma ceae); and, Nannochloris coccoides Naumann
(Division Chiorophyta, Order Chlorococcaies).
Biodiesel was suppiied by Daigety Oils eeds, Newmarket and marine diesa! by Better Boating Co., Reading.

3.2.

Results and Discussion

Growth was calculated as the difference in cell density between the initial (T,) value and that at the end of
the 96 h incubation period (T,). All data was subject to analysis of variance. The values obtained are
shown in Table 1. Mean specific growth rates were also calculated Fable 2).
The growth of all the species tested was reduced by both diesel and biodiesel oils, though each species
shows different levels of response to the fue!s.
Selenasirum capricomufum, the standard OECD test species, was particularly sensitive to marine diesel.
Even the lowest dose (1 g/l) was lethal to this species. Biodiesel, however, was considerably less toxic,
causing only a slight, non-significant, reduction in growth at the lowest dose and still permitting some growth
at the highest dose.
Effects on C. vulgaris, hf. aeruginosa and N. coccoides were somewhat similar. Biodiesel significantly
reduced growth at all concentrations though the effect at the lowest dose rate was relatively small. At the
highest dose rate growth was still measurable except with M. aeruginosa, a blue-green species, which was
killed.
The other blue-green species, A. spiroides showed highly significant growth stimulation by biodiesel at all
dose rates. A similar effect was found with marine diesel at the two lower doses although at 100 g/l diesel
was signiiicantly inhibitory.
These results show clearly that biodiesei is significantly less toxic than marine diesel to a diverse range of
freshwater aigae, although it is toxic to ail but one of the species tested.
The results obtained with S. capricornutum show an extreme sensitivity to the diesel compared to the
majority of the algae tested. On the other hand the magnitude of its response to biodiesel was more
representative. This differential in the response of this species to two toxicants, compared to that of other
freshwater algal species, raises doubts about its suitability as a standard test organism that is supposedly
representative of a range of algal species.

Table 1

The effect of biodiesel and marine diesel on the growth of freshwater algae.
Values are means of 3 replicates with Standard Errors in parentheses.

Species

Growth

Dose
Rate
Control

Chlorella vulgaris

1
100
0

2.58
1.71

(0.27)
(0.23)
1.52 (0.52)

fY3 (0.32)
1.29 (0.40)
-0.40 (0.21)

16.00 (3.20)
1.72 (0.28)
-0.13 (0.05)

12.00 (0.65)
1.75 (0.32)
6.20 (0.02)

11.5 (4.00)

17.80 (7.40)
6.76 (3.20)
0.42 (0.11)

32.1 (8.2)

10

Anabaena

Diesel

4.16 (0.22)

2
20
200
Microcysris
aeruginosa

Biodiesel

1.59 (0.31)

spiroides
1

10.40
8.31

10
100
Selenastrum
capricornutum

3.32 (0.77)

1
10
100
Nannochloris

(3.5)
(3.0)

3.09 (0.50)
0.79 (0.30)

J.32 (0.04)

0.36 (0.25)

-1.33 (0.02)

-0.35 (0.03)

12.8 (0.55)

coccoicies

9.92 (0.50)

10

1.86 (0.12)

2.71 (0.33)
1.37 (0.08)

100

1.01 (0.16)

0.59 (0.05)

Table 2

The effect of biodiesel and marine diesel on specific growth rates of

freshwater algae

Species

Dose
Rate

Specific Growth Rate

g/1
Control
Chlorella vulgaris

Diesel

0.427
0.346
0.324

0.337
0.296
-0.308

1.002
0.479
-0.144

0.930
0.483
-0.286

0.496

2
20
200

Microcystis aeruginosa

El iodiesel

1.173

1
10
100

~kabaena spiroides

1.224

1
10
100

Seienastrum
capricornutum

0
1
10
100

1.265
1.025
0.383

0.471
0.186
0.102

a.148
XI.170
-0.163

0.831
0.449
0.330

0.387
0.239

0.432

1
10
100

Nannochioris coccoides

1.155
1.130
1.076

0.893
0.530

4.

TOXICITY TO FLOATING MACROPHYTES: Lemna minor and Lemna miwra.

4.1.

Materials and Methods

Toxicity to L. minor and L. minfla was determined through a series of modified toxicity tests (ASTM,

1991).
Lemna plants were grown in 15 ml of a prepared culture medium (3 parts JMl and 1 part soil extract)
in 60 mm diameter Petri dishes. Plants from a stock culture were assigned randomly, 6 green fronds to
each dish. The dishes were incubated in a controlled environment room at a temperature of 20 IIC
and a light intensity of 160 9 mol photons Tn.* s.
After an establishment period of 24 hours, oil was added to the dishes to achieve dose rates of I, IO
and 100 g/l. Each dose rate and the controls were replicated three times. The dishes were returned to
the controlled environment room for a further 7 days and the number of green fronds in each dish and
their appearance were recorded daily.
Biodiesel is immiscible with water and, at low dose rates, tends to form discrete globules allowing the
plants to grow around the edges of the test dish without contact with the biodiesel. Attempts to
eliminate this problem, by emulsifying the biodiesel, were made using 5 emulsifiers. These were tested
for toxicity against the blue green alga Microcysfis aeruginosa in a standard 4 day bioassay on
Jaworskis medium (JMI). (See 3. Toxicity to Alga e, Materials and Methods for method) at dose
rates of 0.001, 0.01 and 0.1 g/l. This failed to find a non toxic emulsifier and the approach was
abandoned. In a further attempt to minimise the problem of biodiesel immiscibility, the experiment was
redesigned using 200 ml of growth medium (10% JMI v/v) in 500 ml jars. Approximately half the volume
of medium was rapidly mixed with the appropriate dose using a Waring blender for 30 seconds and
transferred to the test jar. The blender was washed with the remaining 100 ml of medium for 30
seconds. Control (oil free) solutions were treated similarly and oil treatments were prepared in the
sequence lowest to highest dose rates. The blender was cleaned with detergent, thoroughly rinsed and
autoctaved between use for the two oils. Five dose rates 0.0125, 0.125, 1.25, 12.5 and 125 g/l were
used. The minimum dose rate required to give complete surface cover was 12.5 g/l.
After blending, the medium was left for 24 hours before adding the plants which were taken from
laboratory stock cultures grown on the same medium (10% JMl) in the same controlled environment
(temperature 203C i /lC, light intensity 160 p moi photon m s) for at least 8 weeks. Plants were added
to each chamber randomly until all jars contained 3-5 plants each consisting of 3-4 fronds with a total
of 15 fronds in each jar. The composition of fronds/plant in each jar was noted.
Change in plant colour, break-up of plants, destruction of roots and ihe number of fronds, were recorded
daily for 7 days. Every frond that visibly projected beyond the edge of the parent frond was counted
as a se,parate frond. After 7 days a subsample (8 ml) of the medium in each jar was taken from about
half way down the jar and piaced in a 60 mm glass Petri dish. Fresh plants from the stock culture were
added to the dishes, as described above, to give 15 fronds per dish and obsemations and number of
fronds were recorded daiiy for a further 7 days.

4.2 Results and Discussion

Growth was calculated as the mean increase in the number of green fronds in each jar after 7 days.
These values are presented in Tables 3, 4 and 5.
The results of the tests carried out in Petri dishes Fable 3) show clearly that both biodiesel and marine
diesel exhibit toxicity to Lemna minor at similar levels, the effect being non-significant at the lowest dose
rate but increasing markedly with dose. Growth at the highest dose rat6 was only about 35% of that
in the control.

Table 3

Species

The effect of biodiesel and marine diesel on the growth of Lemna minor and Lemna
mix.rta. Values are means of 3 replicates with Standard Errors in parentheses.
Petri dish Experiments.
Dose rate
g/1

Growth (increase in number of green fronds)

Control

L. minor

L. minuta

Cl

Biodiesel

Diesel

7.3 (0.3)

5.7 (1.4)

6.7

(1.2)

10

4.0 (0.6)

3.3

(0.3)

100

2.7 (1.2)

2.3

(0.9)

18.7 (2.2)

14.3

(2.0)

13.0 (1.1)

10

11.0

(0.6)

3.3 (1.3)

100

7.3

(1.2)

0.0

Lsmna minuta, which had a faster growth rate than L. minor in this test, was significantly less affected
by biodiesel than by marine diesel, especially at the highest dose where diesel completely inhibited
growth but biodiesel permitted growth at approximately 40% of growth in control experiments.
i. minuta grew less well in the large jars, only doubling frond numbers in 7 days compared to a threefold increase in the Petri dish experiment (Table 4). The growth of L. minor was the same in both
experiments, even though the growth mediium was considerably more dilute in the jars (10% JMl) then
in the Petri dishes (75% JMl). Considering this change in medium concentration it is, perhaps, not
surprising that both L. minor and L. minufa were more susceptible to the growth-inhibiting effects of both
oiis in the jars than in the Petri dishes. As noted before, however, the effect of both oils was similar on
_1 . minor but diesel was significantly more toxic to L. minuta than biodiesel. indeed, diesel at 1.25 g/l
prevented growth of L. mintia whereas in biodiesel at the same dose this species maintained more than
60% of the growth on the control.

Table 4.

Species

L. minor

L. minuta

The effect of biodiesel and marine diesel on the growth of Lemna minor and Len-m
minuta. Values are means of 3 replicates with Standard Errors in parentheses.
Experiment in jars.

Dose
rate
Q/l

Growth (increase in number of green fronds)

Biodiesel

Control

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

19.7 (0.3)

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

16.3 (3.6)

Diesel

5.7
3.0
0.7
6.0
-1.7

(2.2)
(1.0)
(1.9)
(1.7)
(1.3)

2.7
6.0
-0.7
-6.0
-7.6

(0.9)
(1.5)
(0.9)
(1.1)
(0.9)

11.7
14.0
10.3
9.0
5.3

(2.0)
(0.6)
(2.7)
(1.7)
(3.7)

15.3
10.3
-2.3
-8.7
-11.7

(1.1)
(3.3)
(1.2)
(0.3)
(0.3)

It must be borne in mind that these tests were done using a nutrient medium to support growth of the
plants. The nutrient concentrations used in these experiments are greater than those usually found
under natural conditions in the field. The degree of toxicity exhibited by the test species will be related
to the nutrient status of the receiving waters. Both biodiesel and marine diesel will have a greater impact
on plant health in oiigotrophic (low nutrient) waters than in eutrophic (high) or mesotrophic (medium)
waters. Results from these experiments are more likely to reflect toxicity in mesotrophic or eutrophic
waterbodies. Care should be exercised when extrapolating these data to natural scenarios.
The effect of blending the oil in the nutrient medium before introducing the plants could be said to
simulate the effect of boat propellers. It could be that, by dispersing the oil throughout the solution for
a period, it has allowed the release of water miscible components which may have contributed to the
increased toxicity found in the jar experiment.
This suggestion is supported by the results from the experiment in which plants were grown in nutrient
medium which had been in contact with the oiis for 7 days before planting. (Table 5). The susceptibility
of both species to both oils, but especially to biodiesel, was significantly increased. Thus, whereas 12.5
g/l of biodiesel permitted some growth (c. 30 to 50% of control) when it was introduced to the medium
at about the same time as the plants, the same dose caused some mortality of both species when the
oii had been aged in the medium for 7 days before plants were introduced. Undoubtedly, some
degradation had occurred in this time (see later) and may have increased the toxic response of the
plants. Had the eariier experiment been continued for a funher 7 days, this additional toxicity may have
been seen. However, this effect was not seen in other toxicity tests. An alternative explanation is that
degradation reduced the viscosity of the oil and allowed a film to spread over the water surface, thus
increasing contact with the plant fronds. However, the toxicity of marine diesel, which forms a film on
the water in its raw state, is also increased by ageing. It is, therefore, uniikeiy that fiim formation is a
likely factor.

The magnitude of the toxicity effects found in these tests must be viewed with some caution.
Macrophytes are oft en very variable in their growth, resulting in large standard errors as found here.
Future experiments should involve much larger numbers of replicates.

10

Table 5.

Species

L. minor

L. minuta

The growth measured as the increase in the number of green fronds, of Len-ma
minor and Lemna minuta on 7 day old medium contaminated with biodiesel and
marine diesel. Values are means of 3 replicates with Standard Errors ii
parentheses.

Dose
rate
g/1

Mean average_ growth

(number of green fronds)
Biodiesel

Control

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

5.0 (1.0)

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

11.7 (0.9)

Diesel

3.7
1.3
-0.7
-4.7
-5.3

(0.3)
(1.2)
(0.9)
(0.3)
(0.9)

2.3
0.0
-4.3
-5.3
-10.7

(0.3)
(1.7)
(3.2)
(1.4)
(0.9)

12.0
11.3
6.3
11.0
-8.3

(1.5)
(1.2)
(1.4)
(1.5)
(0.9)

4.0
1.3
-0.7
-5.3
-11 .o

(1.1)
(1.2)
(1.8)
(1.8)
(0.6)

One problem noted here, but applicable in all the experiments, was that even at high dose rates, the
amount of oil to be added to each test unit was very small. For example, even in the large volumes in
the jars, doses of 0.125 g/l require that only 25 ~1 oil to be pipetted into the jar. The accuracy of
pipetting such small volumes, even with automatic micropipettes, is compromised by the viscous nature
of the oils, especially of biodiesel.

11

5.

TOXICITY TO SUBMERGED MACROPHYTES: i?%&a cmadensis and Myriophyllum spicaf~rrr

5.1

Materials and Methods

The toxicity of biodiesel and marine diesel to Elodea canadensis and Myriophyllum spicatum was assessed
using 14 day toxicity tests on individual plants grown in 3 litre glass jars.
Sediment, consisting of 500 g sieved soil under 250 g silver sand overlaid with 300 g washed gravel was
placed in each jar. Healthy single stems (IO cm long) were cut from plants gathered from an outdoor stock
pond. Each stem had a terminal meristem, no branches or roots and all were of a similar weight. Three
stems were weighed and planted in the jar. The jars were filled with 2 litres of ::rater taken from a natural
bore hole source and the initial water level marked on each. The planted jars were randomised and kept
indoors at ambient temperature and light level for 48 hours, after which they were placed in a greenhouse
(25/15OC day/night: 16 h photo period) and oils were applied to the sunace of the water. The dose rates
used were 0.0125, 0.125, 1.25, 12.5 and 125 g/l. Three replications of each dose rate and of controls were
prepared.
Water losses due to evaporation were corrected each day using bore hole water and observations such as
changes in coiour and breakdown of plants were recorded. After 14 days the length and weight of each
plant in each jar was recorded.
A second experiment was established to investigate the toxicity of biodiesel and marine diesel as
contaminant in the hydrosol (hydrosol refers to the sediment component of the experimental system when
fully saturated with water). The experiment was set up as above but the oils were added to the soil before
the macrophytes were planted. The oil and the soil were mixed thoroughly for 5 minutes using a clean giass
rod and taking care to ensure mixing was similar in all the jars. After the oil and soil had been mixed
together, the sand and gravel were added and the macrophyte stems were planted. ?ars were incubated
as above for 14 days.

12

5.2

Results and Discussion

The results presented in Tables 6 to 9 indicate that the growth of the two test species was erratic. The
values for weights of control plants vary by a factor of 2 or 3 between the two experiments and those for
stem length by a factor of 2 for M. spicarum and by 7 for E. canacfensis. Standard errors were frequently
high, sometimes being as large as, or larger than, the mean values. in further experiments, in order to
improve the precision of the data obtained, large numbers of replicates will be required and efforts should
be made to use genetically homogenous plant material. The erratic results may reflect accurately the
situation in natural populations, where some individuals may be less susceptible than others. No figures can
be assigned to predicted population toxicity.
The effect of biodiesel and marine diesel applied to the water surface on the growth
of Elodea canadensis and ylriophyilum spicatum.

Table 6.

Species

Dose

rate
g/1
E. canadensis

0.0000

Growth* (g)
Control

0.0000

-0.09
-0.16
-0.08
0.73
0.18

(0.11)
(0.04)
(0.05)
(0.06)
(0.06)

-0.02
-0.18
0.05
0.11
0.16

(0.04)
(0.03)
(0.13)
(0.07)
(0.03)

0.65
0.25
0.22
0.43
-0.04

(0.14)
(0.45)
(0.44)
(0.12)
(0.37)

0.01
-0.25
-0.40
-0.71
-0.44

(0.11)
(0.06)
(0.01)
(0.29)
(0.32)

0.25 (0.10)

0.0125
0.7250
1.2500
72.5000
125.0000
*

Diesel

0.16 (0.07)

0.0125
0.1250
1.2500
72.5000
125.0000

M. spicatum

Biodiesel

Growth is expressed as increase in fresh weight during 14 days.

Figures in ( ) are standard errors of the means.

Bearing in mind the erratic and imprecise nature of the data it is difficult to draw firm conclusions about
the effects of the oil treatments on the test species. Nonetheless some trends do appear in the data.
The response of M. spicarum to oils applied to the water surface appears to some extent to be similar to
that of other plant species such as algae and Lemna. Growth is relatively unaffected at all dose rates of
biodiesel except the very highest (725 g/I) where it severely inhibits growth expressed as fresh weight
gain or as increase in stem length. The effect of marine diesel is much more pronounced on growth
expressed as weight gain, doses as low as 0.0725 g/l causing virtual cessation of growth while at all
higher doses weight loss occurs, indicating plant death and decay. In contrast, the effect on growth
expressed as increase in stem length is less pronounced. Stem length is probably not a good measure
of plant response to toxicants such as oils as it can be markedly affected by shading caused by the oil
film on the water surface. This would be particularly marked with coloured marine diesel, especially at
the high doses which gave relatively thick surface layers. The effect of this shading increases stem
length, thus masking reduction in growth resulting from toxicity.

13

The effect of biodiesel and marine diesel applied to the water surface on the growth
of Eicdea canadensis and h-fyriophyihm spicatum.

Table 7.

Species

E. canadensis

M. spicarum

Dose
rate
g/1

Growth* (cm)
Control

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

1.76 (2.44)

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

8.50 (1.65)

Biodiesel

Diesel

-1.37
-1 Xl
0.60
2.88
2.54

(1.20)
(1.51)
(2.23)
(1.95)
(2.35)

-0.32
-2.90
1.69
1.90
2.77

(1.9:)
(1.55)
(2.53)
(2.54)
(1.37)

15.78
10.44
10.89
Il.41
5.02

(6.99)
(4.76)
(5.07)
(4.27)
(3.74)

3.39
2.22
1.89
0.67
0.83

(4.55)
(3.42)
(1.10)
(1.84)
(0.87)

Growth is expressed as increase in length during 14 days.

Figures in ( ) are standard errors of the means.

Results with E. canadensis were curious. Very low dose rates of biodiesel or marine diesel appeared to
completely inhibit growth measured as weight change or as stem length increase. In contrast higher
doses caused no discernable effect, other than the expected increase in stem length due to shading
effects.
Application of the ok via the hydrosol had somewhat different effects on E. canadensis Fables 8 and
9). Low doses of biodiesel had a small inibitory effect on growth, both as weight gain and stem length
increase. This effect on plant weight was not measurable at 1.25 g/l and at higher doses plant weights
were significantly higher than control values. Stem lengths were inhibited to a similar extent at all doses
except 12.5 g/l when no significant effect was found. It is likely that this value is a spurious result,
although the standard error is not particularly large.
Marine diesel affected E. canadensis in a more expected and progressive way. Eoth weight and stem
length were unaffected by doses up to 1.25 g/l but thereafter were progressive!y and severely inhibited.
M. spicatum growth was inhibited by both biodiesel and marine diesel applied to the hydrosol (Tables 8
and 9), the data showing an unexpected pat-tern for both weight and stem length. Inhibition was
greatest at the lowest dose, reducing as dose increased until at 12.5 g/l of biodiesei growth was similar
to that in the control. At the highest dose, inhibition was again evident. A similar trend was found with
diesel though the least inhibition was found at 1.25 g/l.

14

The effect of biodiesel and marine diesel applied to the hydrosol on growth of

Table 8.

Eiodea canadensik and Myiophyilum spicatum.

Species

E. canadensis

M. spicatum

Dose
rate
g/t

Growth* (g)
Control

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

0.38 (0.02)

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

0.89 (0.17)

Biodiesel

Diesel

(0.04)
(0.06)
(0.18)
(0.08)
(0.12)

0.38
0.47
0.56
0.17
0.01

(0.06)
(0.06)
(0.12)
(0.12)

-0.26 (0.18)
-0.08 (0.18)
0.14 (0.19)
0.89 (0.42)
0.05 (0.09)

-0.23
0.01
0.68
0.03
-0.20

(0.25)
(0.10)
(0.41)
(0.17)
(0.03)

0.21
0.17
0.43
0.69
0.67

(0.05j

Growth is expressed as increase in fresh weight during 14 days.

Figures in ( ) are standard errors of the means.

Table 9.

The effect of biodiesel and marine diesel applied to the hydrosol on growth of
Efcdea canadensis and h4yriophyDum spicatum.

Species

E. canadensk

M. spica turn

Dose
rate
g/t

Growth* (cm)
Control

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

12.22 (2.46)

0.0000
0.0125
0.1250
1.2500
12.5000
125.0000

19.28 (2.86)

Biodiesei

Diesel

6.80 (1.41)
4.84 (2.57)
8.96 (3.31)
10.50 (1.12)
6.48 (2.04)

13.28 (2.36)
12.54 (1.41)
11.41 (3.08)
8.92 (3.52)
0.47 (1.75)

5.72 (2.39)
8.18 (4.08)
11.24 (4.00)
17.92 (5.00)
5.89 (1.95)

5.23 (3.22)
9.76 (2.97)
15.99 (4.85)
6.73 (1.98)
1.33 (0.73)

Growth is expressed as increase in length during 14 days.

Figures in ( ) are standard errors of the means.

15

Further experiments on the effect of contaminated sediment should concentrate on the effect on root
function. This could be measured by nutrient uptake studies.
As was found with Len-ma and with the microalgae, the effects of the diesel oils varies according to
species, M. spicatum being more tolerant than E. canadensis in general. Also, further differences occur
depending on whether the oil is present on the water surface or in the hydrosol, the former exerting
more toxic effect than the latter. Overall, the results suggest that biodiesel is less toxic to these
submerged plant species, especially M. spicalum , than marine diesel. These differences in susceptibility
may be a factor in altering species balance and diversity in contaminated waters, especially with marine
diesel.
Toxicity tests on macrophytes are of necessity long term, especially if effects on flowering, seed
production and vegetative reproduction are to be assessed. Even in the simple preliminary test reported
here, 14 days was required to obtain measurable effects. During such extended periods there may well
be indirect effects due to the oils. For example, they may have differential effects on diffusion of oxygen
and carbon dioxide into the water and between water and hydrosoil. This would be most likely at the
higher doses where cohesive films of oil are farmed on the water surface or where gross contamination
of sediments occur. Comprehensive detaiied tests need to recognise this and water and sediment pH
and oxygen content should be monitored.
During the experiments it was noted that the leaves of the plants in the jars contaminated with diesel
darkened and turned black. A similar effect occurred in the biodiesel treatments but was generally limited
to the tips of the leaves. Some breakdown of parts of, or whole plants, occurred. This was more
evident in the diesel treatments and when the oil was applied in the hydrosol rather than to the water
surface. There was no distinct dose effect on this symptom.

16

6.

TOXlClTY TO INVERTEBRATES: Daphnia magna, Gammarus pulew and Lymnaea peregm

6.1

Materials and Methods

Acute toxicity to the water flea Daphnia magna ws assessed in a standard (OECD. 1984) acute
immobilisation test using dose rates of 1, 10 and 100 g/l.
Oils were added to 100 ml bore hole water in 250 ml flasks using a micropipette with four replications of
each dose. Treatments and controls were mixed on a rotamix for 1 minute at 100rpm and were incubated
at 20 i 1C and a light intensity of 160 p mol photon m s for 24 hours before introducing invertebrates.
The Daphnia used in these experiments were reared in bore hole water in the controlled environment. Young
(24 h) Daphnia were chosen and randomly assigned to flasks until each flask contained 5 animals. Flasks
were covered with aluminium foil, stoppered with a foam bung and returned to the controlled environment
room.
The number of Daphnia trapped at the water surface or immobilised were counted at 24 and 48 hours.
Immobile is defined as those animals unable to swim within 15 seconds after gentle agitation of the test
chamber . The test was considered valid if < 10% of the Daphnia in the controls were immobiie or trapped
at the water surface.
The results of a preliminary test showed a large increase in immobility between treatments of 1 and 10 g/l
so the test was repeated using 8 dose rates from 0.5 gl* increasing by a factor of 0.6 up to 13.422 gIW.
Gammarus puiex and Lymnaea peregra were collected from a local stream and acclimatised in tanks of
borehole water for at least 48 hours.
Large (500 ml) glass jars were filled with 709 silver sand and 25Oml bore hole water. immature Gammarus
of similar sizes and colour were chosen from the sample population and randomly assigned to jars until all
the jars contained 5 animals. The jars were illuminated at an intensity of 125 p mol photon me2 se in a 8:16
hour light:dark regime at ambient temperature (21 :l lC day/night).
After 24 hours, lgl-, 10 gl-, or 100 gl doses of biodiesel or marine diesel were applied by micropipette
to the surface of the water in the jars. Four replications of each dose rate and control were prepared, using
a random assignment of dose rates to jars.
At 24, 48, 72 and 96 hours after exposure to oii, the number of dead or affected animals were recorded.
Death and immobiiisation were defined as a lack of movement and a lack of response to gentle prodding.
Other observations such as erratic swimming, excitability, loss of reflex, disclouration, cessation of burrowing
and behavioural changes were also recorded. The test was considered to be valid if < 10% of animals in the
controls were dead after 96 hours. Acute toxicity to Lymnaea peregra was assessed in the same way.

17

Results and Discussion

6.2

The values of mean percentage immobilisation of Daphnia magna at each dose rate (Table 10) and of mean
percentage death Fable 11) show cleariy that marine diesel is highly tokic to D. magna, all animals being
immobilised at the lowest dose (0.5 g/l-) within 24 h. In contrast, although biodiesel was toxic, complete
immobility was only recorded at the highest dose (i3.4 g/l) after 48 hours. At lower doses between 40 and
55% of the population tested showed no discernible effect of treatment. In all cases where immobilisation
occurred, it was followed by death, as determined by observation of heart beat. It is not known whether
transfer of immobiiised but living animals to clean water would allow recovery.

Table IO.

The effect of biodiesel and marine diesel on Daphnia magna.

immobiiisation (%)

Dose
rate
9/l

24h
Control

Biodiesel

48h
Diesel

Control

Biodiesel

Diesel

35 (5.0)
95 (5.0)
100 (5.0)

100 (0.0)
100 (0.0)
100 (0.0)

45 (5.0)
50 (5.8)
25 (9.6)
30 (5.8)
50 (5.8)
50 (5.8)
60 (8.2)
100 (0.0)

100
100
100
100
100
100
100
100

Experiment I.
0
1
10
100

10 (5.2)

7 (4.9)
20 (8.2)
80 (8.2)
85 (5.0)

100 (0.0)
100 (0.0)
100 (0.0)

20
35
25
30
40
25
55
65

100
100
100
100
100
100
100
100

Experiment 2.
0.000
0.500
0.800
1.280
2.048
3.277
5.243
8.389
13.422

3 (2.6)

2 (1.8)
(0.0)
(5.0)
(9.6)
(5.8)
(0.0)
(9.6)
(9.6)
(5.0)

(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)

(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)

Data are mean ?L of animals immobilized with standard errors in ( ).

G. pulex was more sensitive to the biodiese! than D. magna, a high proportion being killed within 24 hours
of exposure at reiatively low doses (Tabis 11). Even the lowest dose tested (1 g/r) caused 5046 mortality
after 72 hours, though there was no increase thereafter. As with D. magr;a, marine diesel was highly toxic,
The lowest dose causing 100% mortaiity in 24 hours.

18

Effect of biodiesel and marine diesel on Gamman~s pulex.

Table 11.

Mortality (%)

Dose
rate
g/l
Control
0
1

48h

24h

Biodiesel

Diesel

(0.0)

25 (0.0)
95 (0.0)
100 (0.0)

100 (0.0)
100 (0.0)
100 (0.0)

72h
0

Diesel
100 (0.0)
100 (0.0)
100 (0.0)

96h
5 (5.0)

5 (5.0)
50 (23.8)
100 (0.0)
100 (0.0)

1
10
100

Biodiesel

0 (0.0)
10 (5.8)
65 (5.0)
90 (5.8)

10
100

Control

50 (23.8)
100 (0.0)
100 (0.0)

100 (0.0)
100 (0.0)
100 (0.0)

100 (0.0)
100 (0.0)
100 (0.0)

Figures in ( ) are standard errors of the means.

Similar effects of marine diesel were found with L. peregra (Table 12), with 95% mortality at 1 g/I- in 24
hours and 100% mortality at 72 hours. Biodiesel, in contrast, exerted toxic effects only at 100 g/l-; mortality
increasing from 15 % at 43 hours to 90% at 96 hours.
Table 12.

Effect of biodiesel and marine diesel on Lyrnnaea peregra

Dose
rate
g/f

Mortality (A)
24h
Control

Biodiesel

48h
Diesel

0 (0.0)
90 (10.0)
95 (5.0)
95 (5.0)

0 (0.0)
0 (0.0)
15 (9.6)

96h

72h
0

1
10
100

Biodiesel

Diesel

5 (5.0)
0 (0.0)
0 (0.0)
0 (0.0)

1
10
100

Control

5 (5.0)

95 (5.0)
95 (5.0)
100 (0.0)

~~

5 (5.0)
0 (0.0)
0 (0.0)
50 (5.8)

100 (0.0)
100 (0.0)
100 (0.0)

5 (5.0)
9 (0.0)
90 (10.0)

Figures in ( ) are standard errors of the means.

19

100 (0.0)
100 (0.0)
100 (o.oj

Marine diesei was highly toxic to all species at all COnCentratiOnS, the effects becoming apparent very
quickly. Biodiesel was much less toxic especially at the lowest dose, representative of a small spillage.
There were, as with plants, different responses from different species. L. peregra was very tolerant of
biodiesel whereas G. pulex and D. magna were tolerant Only Of low doses. Again, as was found for plants,
it seems that pollution of water with biodiesel may cause some shift in species balance in the habitat.

20

7.

TOXICITY IN AQUATIC MICROCOSMS

7.1

Materials and Methods

Microcosms were established in small (0.6 x 0.3 x 0.3 m) aquaria containing a basal layer of 6 kg loam soil,
overlaid with 3 kg silver sand and then 3 kg of washed gravel. Tanks were filled with 40 I of water from a
bore hole and aerated with an air diffusing stone at 4 1 air minute-.
Stems of Elodea canadensis and Myriophyllum spicatum were carefuily selected, as in section 5, to have
stem weights of 26i 1 g or 82 1 g respectively. Plants were weighed accurately and they were planted into
the hydrosol. Twenty plants of each species were planted in separate but adjacent blocks so that not more
than 25% of the water body was occupied by plants.
Planted tanks were incubated under lights at 125 p mol photons m s- with an 8:16 hours light:dark regime
for 6 weeks before fish and invertebrates were introduced. All the animals had been kept in stock tanks,
under the same conditions as the microcosms, for at least three weeks before being used in the experiment.
Daphnia magna were placed in the microcosms in small fine-mesh enclosures to prevent predation by the
fish. Each enclosure containing 20 young (24 hr) animals was maintained at a depth of 20 cm in the water.
Lymnaea peregra were added directly to the aquaria such that each contained 3 large (>3.5 cm long shells),
5 medium (2 to 3 5 cm) and 2 small (~2 cm) snails. The collective weight of the 10 snails added to each
tank was recorded.
Fingerling (3 inch) Rainbow trout (Oncorhynchus mykiss ) were weighed and three placed in each tmk.
The populated microcosms were then left to stabilize for one week before adding the oils. Throughout the
experiment the trout in each tank were fed 1.5 g of proprietary trout peilets each day and the evaporative
losses from the tanks made up with fresh borehole water.
Oils were added to the tanks using micropipettes to give dose rates of 0.002, 0.02 and 0.2 litres m. These
dose rates were calculated from the assumption that spillage of the complete contents of the average
pleasure boat fuel tank (50 gallons or 227 litres) would be restricted to an area of 1000 m*. This worst case
scenario would produce 0.2 I m.
Due to constraints of space, available tanks and time, the doses were not replicated but three control +anks
were set up. Microcosms were observed daily for 3 weeks and any dead fish removed using a separate net
for each tank. While every care was taken in this operation it is unavoidable to trap some of the surface oii
on the net, especially at the higher doses. The use of very long forceps can minimise this problem but even
then some oil is removed from the tank at each operation. Dissolved oxygen, pH and water temperature
were monitored throughout the experiment.

21

7.2

Resutts and Discussion

Ambient temperatures during the experimental period were very high, frequently reaching 27-29C. As a
result, water temperatures rose rapidiy (from the optimum 18C) to 25OC and remained at that level
throughout the experiment. Although the elevated temperature had little effect on the dissolved oxygen
content of the water, it affected plant respiration and, as a consequence, pH rose to levels around 8.8.
Before the fish and animals were added to the tanks the pH was brought down to c. 7.0 by adding 0.1 m
HCl and 5.25 mol mJ HEPES buffer to each tank. Subsequently pH values stayed relatively constant
berween 7 and 8 (Appendix 1).
The increase in water temperature adversely affected all the members of the microcosm community. Plants
became sickly and failed to grow in the controls as well as in the treatments. Similarly, invertebrate
behaviour was atypical and the mortality rate in the controls was high enough (> 10%) to invalidate the
tests. Fish were also affected and by two days after their introduction were gathering by the air-stones or
gulping air at the surface even though the dissolved oxygen content was close to 100% saturation. The
gulping of air at the surface may have led to intake of oil, especially marine diesel (see comments on oil
behaviour below), and contamination of the gills. The fish showed progressive darkening, a typical symptom
of stress, in both control and treated tanks. In the control tanks about half the fish died 5 or 6 days after
the treatment date, the remaining fish surviving until at least 9 days after treatment.
This test is invalid as a long term microcosm study but it is worth recording some observations of fish
behaviour, as these do indicate effects induced by the oils.
In the two iowest dose rates of biodiesel fish were initially little affected, showing only the stress symptoms
noted above. Ereathing and fin movements were no different to those in fish remaining in very large stock
tanks where conditions were optimal for growth. However, on day 2 after treatment all these fish died. Fish
in the highest biodiesel treatment all died 1 day after treatment, after showing erratic breathing, cessation
of fin movement and hovering at the water surface.
Low dose rates of marine diesel caused similar effects to biodiesel. Fish behaviour was normal on the first
day after treatment but on day 2 all the fish but one died. The survivor (at 0.02 I m) remained stationary
on the sediment until day 9 when it came to the surface, breathing erratically and showing severe loss of
balance. Fish exposed to the highest dose of diesel showed signs of stress within one or two hours of
treatment. They began to swim with their bodies in the horizontal plane, with complete loss of balance and
gulping air at the surface continuously. Toward the end of the first 24 hours symptoms were extreme loss
of balance and of coordination of movement. One fish was showing frequent spasms every 5 seconds. ,411
fish had died at the end of the first day after treatment.
All the fish were weighed at death and the weight losses are shown in Table 13. It is apparent that the
marine diesel treatment is associated with much greater weight loss than either the biodiesel treatment or
control which showed similar but lower losses. This would indicate that, as in all other tests, the marine
diesel is considerably more toxic than biodiesel. This is supported by the appearance of much greater
stress symptoms, such as loss of balance, erratic breathing and development of spasms, in fish in the
marine diesel treatments.

22

Table 13.

The mean loss of weight of fish in aquatic microcosms contaminated with biodiesel
and marine diesel oil.

Average Loss of Weight of Fish (g)

Dose Rate g/I
0.002
0.020
0.200

Control

Biodiesel

Diesel

7.57
3.92
5.10

4.71
8.11
3.12

8.27
13.36
28.72

The toxicity of biodiesel to fish and other members of microcosm populations needs to be established
properly in valid tests. This will require some modifications of the method used in this preliminary study.
Larger tanks, with at least 1 m3 capacity, or smaller fish species should be used. Suitable species would
include 3-spined stickleback (Gasterosteus aculeatus) or fat-head minnow (Pimephales promeks). Fish
stocking rates should be below 50 g m3. Further, some provision for water-cooling should be made or tests
restricted to periods when ambient temperatures do not exceed 78C. Acute toxicity can of course be
determined with fish kept in isolation (as in OECD guideiines 202, 1992 and 204, 1984). However, toxicants
which are immiscible in water, such as biodiesel and marine diesel are probiematic in such tests. Dispersal
of the oils with emulsifiers may help hut this will necessitate screening to find non-toxic emulsifiers. The
different behaviours of the two oils when added to water are important. Marine diesel spreads rapidly
forming a thin cohesive siick on the surface. At the lowest dose the slick was unable to cover the whoie
water surface but broke up into discrete isiands. Some of these became trapped at the water/glass
interface, this being aided by the water turbulence caused by forced aeration. At the remaining doses the
slick covered the entire surface. Biodiesel, in marked contrast, formed discrete globules which moved
around the surface with water currents. Many of these were trapped in the meniscus at the water/glass
interface and often coalesced to form larger aggregates. A significant proportion of the biodiesel, after 7
days, had visibly moved down into the water column and formed a white waxy deposit on surfaces of plants
and gravel. Diesel was not seen to move into the water column in this way although the sand layer of the
sediment became discoloured at the highest diesel dose.

23

BIODEGRADATION AND FATE

8.1

Materials and Methods

The microcosms described in Section 7 were also used to determine the biodegradation and fate of the oils
in the aquatic ecosystem. Samples of water, plant material and sediment were taken from each microcosm
at regular intervals.
Water samples (5.0 ml) taken using a micropipette from a depth of 10 cm at each of 3 sites in each
aquarium, were combined into one bulk sample. The same sites were sampled on each sampling date.
Plant samples were obtained from E. canadensis by cutting off the terminal 5 cm from one plant in each
microcosm.
Sediment was sampled using a 25 ml wide-bore glass pipette inserted below the sand and gravel layers and
drawing up the suspension of soil. Samples were taken from 3 sites equidistant along the centre of the
length of the tank and combined into one bulk sample. Samples were taken from random sites on each
occasion. Samples were taken from each microcosm 1, 7, 14 and 21 days after application of the
treatments. Additional samples for analysis of degradation products were taken from the 0.2 I m2 treatments
only on days 3 and 5.
Plant samples were submerged in concentrated HCI for 2 minutes, to remove surface debris and epiphytes,
and then washed gently in distilled water. The washed plant material was ground in a mortar with 1 g of
acid washed sand and 15 ml of ethyl acetate. The slurry was centrifuged at 4000 g for 10 minutes and the
supernatant decanted into McCartney bottles, granular sodium sulphate was added to dessicate the solution
until no further ciumps formed and the dried solutions were stored at 4C until analysed.
Sediment samples and water samples were mixed with 15 ml ethyl acetate and shaken at 60 rpm for 24
hours at room temperature. Thet extracts were allowed to separate for 30 minutes and the ethyl acetate
supernatants transferred to vials with added sodium sulphate granules to dessicate and stored at 4C until
anaiysed.
All ethyl acetate extracts were analysed by Gas Chromatography/Mass Spectrometry (GC/MS) after
concentration by evaporation in a stream of nitrogen at room temperature. Details of the equipment and
operating conditions are given in Appendix 4. Pure samples of both oils were also analysed. After
inspection of preliminary ion chromatograms certain ions were selected as suitable for indicating degradation
of the parer,! oils. Those selected as representative of the methyl esters and biodiesel were ions with
masses m/z - 74 and m/z - 264. The former is the largest ion characteristic of all unsaturated straight chain
methyl esters (C,,,) in biodiesei. Subsequent analyses showed that many peaks on the 74 chromatogram
were not from a biodiesel methyl ester but were contaminants, possibly from plastic tubing used during
concentration of samples before analysis. Consequently, the fate and degradation of biodiesel was tracked
using only 264 ion chromatograms since 264 is known to come only from the C,,, methyl ester in biodiesel.
The ions initially selected as representative of marine diesel were m/z - 91 and m/z - 57. The former is
characteristic of all benzene-containing compounds and the latter of the homologous series of straight chain
aliphatic hydrocarbons. Subsequent analysis showed that several isomeric compounds repeatedly appeared
on the chromatograms and confused the analysis. As the straight chain saturated hydrocarbon region of
marine diesel peaks at hexadecane (C,,) its molecular ion, m/z - 226 was tracked in the samples instead
of 57 and 91.

The ion chromatograms produced show an absolute value for peak intensity, the values of each peak being
normaiised with respect to the highest peak (- 100%). Thus, changes in absolute value for the selected
24

peaks can be monitored throughout the incubation period and indicate the rate of partition of the parent oil
into different components of the microcosm and give some indication of degradation.

25

8.2

Results and Discussion

Analysis of the biodiesel oil showed that the major methyl esters present were (Cl 6:10, Cl 8:10, Cl 8:2, Cl&3
and C22:l) which a library search identified as hexadecanoic ((X6:10), octadecanoic (Cl&lo),
octadecadienoic (Cl8:2), octadecatrienoic (Cl8:3) and erucic acids (C2.21). The major straight chain
saturated hydrocarbons in marine diesel were simiiariy identified as pentadecane, hexadecane and
heptadecane. (Appendix 3.2).
At one stage in this experiment airstones rose to the water surface where they caused some minor
splashing. This contaminated a neighbouring control tank which then gave chromatograms with measurable
amounts of m/z - 26-4. This was not used further in the analyses. Airstones were then fixed in position to
prevent recurrence of this probiem.
The absolute values for the selected tracking ions in the different mesocosm components are shown in Table
14. The results for biodiesel and marine diesel cannot be compared quantitatively as the values used refer
to different ions. Values for an individual oil can be compared and do indicate relative concentrations in the
different microcosm components. However, the sampling regime used did not account for the oil floating
on the surface of the microcosm and, so, the results cannot be used to indicate the total extent of
degradation of the added oil, merely that degradation is or is not occurring. Analysis to accurate!y quantify
degradation would require many more samples, and commensurate large increases in analytical time and
cost. This was not possible within the confines of this preliminary project.

Table 14.

Partition of biodiesel and marine diesel into aquatic microcosm components.

Dose

Time*

rate

(days)

0.002

0.02

0.20

t
t

Biodiesel (m/z - 264)+

Water

Diesel (m/z - 226)+

Plant Hydrosol

Water

Plant

Hydrosol
869

7
14
21

293
244
420
ND

24412
430
500
418

1756
193
406
NT

412
78
574
455

1560
578
47
71

1
7
14
21

200786
2641
404
4

23740
2349
2284
ND

1671168
12206
6169
867

1191

143
306
159

883
NT
536
1294

2251
754
439

1
3
5
7
14
21

1892966
4085453
1022310
24178
ND
ND

162222
716145
344064
209644
14642
2989

4074291
3629056
2638971
233226
ND
ND

48256
1188
2788
519
141
43

6487
11813
94413
4018
2335
4500

75694
12749
29819
26804
555 1
7324

31
99
319
3594

Time is days after treatment

Values given are absolute values; ND is not detected; NT is not tested.

The most obvious feature of ihe results is that both oils very rapidly permeate the whole microcosm.
Significant amounts are found in the water column, on plants and in the sediment only 1 day after
application to the water surface. This is almost certainly largely due to the disturbance caused at the water
26

surface and in the water column by the forced aeration of the system. In the case of biodiesel, the observed
movement of globules onto surfaces where they deposited as a white, waxy, residue (see Section 7) is also
a factor. This was only noticed visually after 7 days but undoubtedly occurred earlier. It would be
reasonable to expect this effect to be greatest at the highest dose rate and, indeed, the very high absolute
values seen in the water column, on plant surfaces and on the sediment might support this.
The second notable feature of the results is the failure to detect biodiesel in most of the samples of the water
column or sediments in the later stages of the experiment. This contrasts with the results for diesel which
was detectable in significant quantities at all sample dates, indicating a slower degradation rate. Diesel
appeared to be particularly recalcitrant in the sediment, as was biodiesel on the plant surface. In the case
of biodiesel, the slow loss from plant surfaces is probably related to the formation of white waxy deposit.
A summary of the persistence of the total oils present in the water column, on plants and in the sediment
is shown in Table 1.5. These figures exclude that part of the added oil which floats on the water surface and,
as stated before, it is not possible to draw direct comparisons between values for biodiesel and diesel as
different ions have been measured.

Table 15.

Time
(days)
l - 7
8 -14
15 -21

Persistence of biodiesel and marine diesel in microcosms. (The data are percentages
of the total ion detected)

Biodiesel/dose rate I me2

Diesel/dose rate I m

0.002

0.02

0.20

0.002

0.02

0.20

94.00
4.60
1.40

99.50
0.46
0.04

97.48
2.50
0.02

70.1
13.1
16.8

61.2
12.2
26.6

94.1
2.4
3.5

These data indicate that the tracking ion for biodiesel enters the microcosm from the surface giobules more
quickly than the diesel ion enters from the surface slick. Equally, the data show that the biodiesei appears
to disappear (degrade) more quickly than the marine diesel.

27

9.

GENERAL CONCLUSlONS

It is important to bear in mind that although, whenever possible, standard toxicity tests have been used, the
constraints of time and resource imposed in this project mean that the results must be regarded with
caution. They represent only a preliminary and superficial survey of the toxicity of biodiesel compared to
that of marine diesel. Furthermore, the adverse environmental conditions that affected the microcosm
experiments detract from the value of data obtained in these experiments. The data for partition and
degradation of the oils will be least affected, the elevated water temperatures .accelerating decomposition
and, probably, being responsible for deposition of biodiesel as white waxy deposits on plant surfaces.
Nonetheless, it looks quite probable that biodiesel does in fact enter the water body more quickly than diesel
from surface deposits. This, along with the globular distribution of the surface biodiese! on the water
surface, will result in less interference with oxygen diffusion into the water, and with surface breathing or
moving invertebrates, than the uniform slicks produced by marine diesel.
Rapid entry is associated with more rapid disappearance from the system by biodiesel than marine diesel.
As the biodiesel appears to form few or no intermediate compounds during degradation, but rapidly to
proceed to CO,, it would again seem to have environmental advantage over the more persistent marine
diesel. This may be offset, albeit temporarily, by an increased biological oxygen demand.
This environmental advantage is strengthened by the significantly lower degree of toxicity of biodiesel
towards most of the algae, macrophytes and animals tested. However, it does have some toxic effect and
this may be enough to allow shifts in balance and diversity of aquatic species, especially where
contamination is severe.
Nonetheless, the environmental advantages which seem to be identified in this preliminary examination are
sufficient .o warrant a more detailed and comprehensive examination and an analysis of the potential to use
biodiesel as a fuel for boats on inland waterways, especially those with recognised conservation value.

10.

REFERENCES

ASTM (1991) Standard Guide for Conducting Static Toxicity Tests with Lemna gibba G3 E 1415 - 91
ASTM (1993) Standards on Aquatic Toxicology and Hazard Evaluation (7,093) 03978-80(1993) Practice for
Algal Growth Potential Testing with Seienasfzrm caprkomlrtum 79.
OECD (1984) Algal Grow&h Inhibition Test, Guideline 207
OECD (1984) Daphnia sp. Acute lmmobiiisation Test, Guideline 2M
OECD (1984) Fish, Prolonged Toxicity Test, Guideline 204
OECD (1992) Fish, Acute Toxicity Test, Guioeiine 203
THOMPSON, A.S.; RHODES, J.C. & PEITMAN, 1. (1988) Culture Collection of Algae and Protozoa Catalogue of Strains. Published by CCAP, Cumbria, UK ISBN 1 871105 0 3

29

APPENDIX 2.

Biodiesel Spectra and Ion Chromatograms

1.

Biodiesel Spectrum showing the peaks of the major methyl esters.

2.

Ion Chromatogram for 1% biodiesel (centre graph) shows that the ion with a mass 74
peaks at SCAN 850 but also peaks at SCAN 720, emphasising the confusion
encountered during analysis.

3.

An example of an Ion Chromatogram produced from the analysis of a sample taken from
a microcosm contaminated with biodiesel.

4.

An example of an Ion Chromatogram produced from the analysis of a sample taken from
a control microcosm.

APPENDIX 3. Diesel Spectra and Ion Chromatograms

1.

Diesel Spectrum.

2.

Ion Chromatogram for 1% Diesei showing ions with masses 57 and 31.

3.

An example of an Ion Chromatogram produced from the analysis of a sample taken from a
microccsm contaminated with marine diesel oii.

40

1.

Biodiesel

SSSULTS

OF ?3M SSARCH

USING

T-5: wilevnbs

I_,iER_B"V
^_._

RT=27:45
?.un=C310005
ScaE=Ii.81
(Su5)
'00%=954368 ADC Eass %nqe=<O-456
25 2uc 94
Compacted Si?.? +"I
o.&LJ 1% S;CDIKSSL IN 2TCAC
13:40
3-1
A\__.

Rel.

Mol.

v. c .
(Sim) (Same) -?i
_78253
97
23 2
77337
91
296
_31 0
46227
70
'-2
52276
_i
59
34
321
113098
^T
77342 z>
296
76740 :8
268
48178
13
322
246
1C1089
12
-.
128
5251
62690
1,
490
25672
252
9
41044
280
9
41045
5
280
1 AYA=
156
._ -4
6
9a_-...
1 Q;g
126
6
::'
A&D
11380
6
224
29648
6
7759
140
6
154
84449
6
-'7
2918
i__
6
?
26225
L_i 0
6
7863
I40
4
i72364
-i-2
4
_--r_
I!0
13oi2
4

sa-?;
___a1

o_Emla

Name
&
* .
C23 ii44 02
~~-Do~~s~xcIc zcia, methyl ester, (Z)CL9 E36 02 9-Gcttdecenoic acid (Z)-, me:hvl ester
C20 ii,38 02 Cyciopropanedecanoic acid, 2-hesvl-, methvl e
13-Doccsenoic acid, methyl ester; (Z)- C23 H44 02
Cl?0 519 N C3
8,9-3imetic~y-2-~henyl-5,6-SihydroDvrro1~~2
9-Gctadecenoic acid, methyl ester, (giCi9 I-I36 0.2
Cl7 ii32 02
9-Fiexadesenoic acid, methyl eszez, (Z)c23 246
IL-Tzicasene
C12 'r;23 3R SROI+OCKi,ODCDSCP.N75
Cl :1:2 02 *Met:hacrvlic acid, propyl esrez
C35 970
17-?enLat=Lacontene
C18 536 3-Occadecene,
(Iz)c20 540 Cyclotetzadecane, 1, 7,11-~=i_~e=~~yl-i-(l-_~e~~~~;~=~
_--c2 0 F40
Cyclohesane, I-(1,5-~ime=~ylhe~.~yl) -<-(4-methyl?e
C'i
A_ El8 0 ?,Ei:xiL CYCXP3JTSNOIE
Cl1 218 03
(')-(1S,3S)-Met~yl(3-acety~-~,2-~~met~~~~c~c~c,
CL0 E20 0 CTS-?->~~NT~>J-9-0~
1,2-dimet~yl-j-se~~=yl-4_3rocvlCl6 ,u.32
Cvc lohexane
-_
c9 H16 0 .5-None--4-dne
CL1 522
__ :?vlcvlcspn~a-L=
(+j-(1s ,2?.)-1-3u:yl- 2-e.CS 915 Cyclopentane, l,l,3-trime:hyll
C15 .F30
~~F?IACT,n_Nz-C
cio "20 CycLopentane, 1,2-dimet~yl-3-(I--et~~~et~y~)Cl0 X16 0 E-Clt;al
8-Xoner,cic acid, methyl ester
c:o EL8 52

2.

75743
75740
36067
75744
36071
76791
75742
45257
79331
58925
23082
52293
78609
65317
L-1407
76iO7
78076
71839
79780
48467
7SiOa
78882
76444
72783
23-33

Diesel

90
92
04
93
93
9 1
91
89
a9
89
89
89

EG
66
66
66
62
62
62
62
6C
60
35
35
25

79
74
70
66
66
50
60
5s
56
56
56
56
56
39
39
2"
_Y
36
36
36
36
35
35
11
li
7

226
225
254
226
254
394
226
310
-Cl.3uo
422
296
352
-n
;c 0
604
252
240
338

28

294
32L_
240
408
254
156
r9a

Cl5
Cl6
Cl8
Cl6
Cl8
c2a
Cl6
c22
C36
c30
c21
c25
C27
C43
c20
Cl-i
C24

234
E34
B3a
x.74
X.38
~58
234
E46
ti7r
H62
244
E52
s56
E98
HZ2
I<36
~50

Ci4 i-:30
C28 258
C23 E48
Cl7 E:.36
c 2 9 ki60
Cl8 E38
Cl1 E24
CL4 x30

Eexadecane
Hexz6ecane
:-!exatincane, 7, C-ei,re-_h ylFiexadecsne
2-T.=-~vlfie=Laciecane,
_-*._
Octacosane
E-Lexadecane
Docosane
Eexztriaconrziie
Triacantane
Tt e 3s i c 3 s an e
Fc77zrcsan2
._._.__I
:< e 2 t acosane
Tri-_-=trac3nt2?l2
"siccsxle
Hez:zdeczle
Tetraccsane
ma--2tiecane
_-__
Oczacosane
T-p-sane
_-__.A
EesczSecane

Xonaczsana
Oc-,adecane
Derzne, 2-methylDo&cane, c-< 5-~imetkiyl-

47

APPENDIX 5.

Mass Spectrometer and Gas Chromatography Instuments.

GAS CHROMATOGRAPHY INSTRUMENT

COLUMN
film thickness
phase

IONlSlNG VOLTAGE

: Carlo Erba hlega

: 25m x 0.22m
: 0.25p
: 3PX70 (specifically apprcpriate for far,y
acid methyl esters)
: spiit/splitless
: Heiium
: 0.5 kg cm-
:7OC for 1 min, upto 240C a: 6C per min,
staying a: 240C for 30 min
: 70 electron volts

MASS SPECTROMETER
Source Temperature
Interface Temperature
SCAN
interscan Time

: Kratos MSeORFA Medium Easolution

: 200C
:
250C
_
: 500 daltons 0 40 daltons at :s per decade
: 0.25

INJECTION MODE
CARRIER GAS
PRESSURE
GC PROGRAMME

APPENDIX 6. List of Suppliers

Giodiesel Oil

Imported from Italy, supplied by Mr. Stephen TUG, Dalgety Oiiseeds Dalgety
Agricultura Ltd., Cheveley House, Fordham Road, Newmarke?, Suffolk CB8 7AH.I

Marine Diesel Oil

Supplied by Better Boating Co., Caversham, Zeading, Berks.

Chemicals

HEPES buffer supplied by Sigma Chemical Co. Ltd., Fancy road, Poole, Dorset
BH17 7BR.
Granular sodium suiphate suppiied by BDH Laboratory Supplies, Merck Ltd., Hunter
Boulevard, Luttemorth, Leicestershire LEI 7 4XN.
Hydrochloric acid and Ethyl acetate supplied by Fisons Scientific Equipment,
Bishop Meadow Road, Loughborough, Leicestershire LE11 ORG.